Renal epithelial cell plasma membranes are divided into two domains whose distinct biochemical compositions reflect their individual roles. This polarity is required for vectorial solute and fluid transport in the kidney. To achieve this asymmetry, an epithelial cell must be able to establish distinct surface domains and to maintain these domains' distinct identities. The generation and maintenance of epithelial polarity is dependent upon the formation of contacts between neighboring cells, which initiates signals that activate the assembly of intercellular junctions and their associated polarization machinery. We and others have shown that adenosine monophosphate-stimulated protein kinase (AMPK) plays a key role in the formation of intercellular junctions and in the establishment of polarity. AMPK is a cellular energy sensor activated by intracellular ATP depletion that helps to protect tissues from the consequences of energy deprivation. AMPK governs numerous pathways that modulate cell growth and metabolism. Extracellular calcium plays a critical and complex role in inducing junction assembly, and we find that the calcium sensing receptor (CaSR) contributes to calcium- dependent junction assembly. We have also found that initiation of junction formation leads to inhibition of the GSK3 kinase, which regulates a number of cell differentiation and polarization signaling pathways. We wish to understand the roles that AMPK, GSK3, the CaSR and other regulatory molecules play in the establishment and preservation of epithelial polarity under normal circumstances and in the face of pathological perturbations such as energy deprivation. We propose to determine 1) how modulation of the activities of AMPK, GSK3 and the CaSR initiate tight junction formation and influence tight junction protein expression; 2) how the AMPK, GSK3 and CaSR pathways influence established junctions in intact epithelia; and 3) how loss of AMPK activity affects renal function and the susceptibility of renal tubular epithelial cells to ischemic damage. Through these studies, we will identify new regulatory targets and control points in the complex process of epithelial polarization. We will also invesigate cellular pathways involved in acute kidney injury and possible therapeutic interventions that may mitigate its severity.